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Technical Abstract: Variability at microsatellite loci has been used widely to infer the extent of genetic diversity among related plant taxa. However, typically, only the most polymorphic loci in the genome were analyzed that may result in a biased, and generally overestimated picture of genome-wide microsatellite diversity. Recently, the genomes of two cucumber (Cucumis sativus L., 2n = 2x = 14, genome size ~367 Mbp) inbred lines, the North American pickling type Gy14, and the Chinese Long fresh market type 9930, were sequenced, which provided a valuable tool to accurately estimate genetic variation of microsatellite sequences. In this study, microsatellite diversity between 9930 and G14 was investigated based on analysis of whole-genome sequence data. Bioinformatics tools were used to detect perfect di-, tri-, tetra-, penta- and hexanucleotide repeats in 203 Mbp assembled Gy14 genome sequences and, for each repeat, a tract of approximately 1kb of flanking genomic sequences was used to find homologous repeat sequences in the 9930 genome. A total of 671,273 common microsatellites with =3 repeat units and a minimum total length of 6 bp were detected. Of these, 621,088 (92.5%) were monomorphic and 50,185 (7.5%) were polymorphic, and in general, the level of polymorphism increased with the number of repeat units in the microsatellite. Because of the large dataset analyzed and the fact that no prior marker selection was performed, our estimate of 7.5% diversity between Gy14 and 9930 likely represents an unbiased picture of genome-wide microsatellite variability in cultivated cucumber.